Application of tomato hsfa1a gene in improving plant autophagosome activity and drought resistance
An autophagosome, gene technology, applied in applications, plant products, genetic engineering, etc., can solve problems such as reduced heat resistance and reduced expression
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Embodiment 1
[0085] Cloning and vector construction of tomato HsfA1a gene
[0086] 1. Tomato Total RNA Extraction
[0087] Adopt Tiangen Plant total RNA extraction kit to extract the total RNA of young tomato leaves, the steps are:
[0088] (1) Take 0.1g of leaves and grind them in liquid nitrogen, add 1mL of lysate, and then process them with a homogenizer;
[0089] (2) Place the homogenized sample at 15-30°C for 5 minutes to completely separate the nucleic acid-protein complex;
[0090] (3) Centrifuge at 12,000 rpm for 5 minutes at 4°C, remove the supernatant, and transfer to a new RNase-free centrifuge tube.
[0091] (4) Add 200 μL of chloroform, cover the tube cap, shake vigorously for 15 seconds, and place at room temperature for 3 minutes;
[0092] (5) Centrifuge at 12,000rpm for 10min at 4°C. The sample will be divided into three layers: a yellow organic phase, an intermediate layer and a colorless aqueous phase. RNA is mainly in the aqueous phase, and the volume of the aqueous p...
Embodiment 2
[0107] Construction of Tomato HsfA1a Gene Overexpression Plants
[0108] 1 Cultivate sterile vaccine
[0109] Soak tomato seeds in tap water (or use a shaker at 28°C 200r / min) for 6-8h, then sterilize with 75% alcohol for 30sec, then sterilize in 10% NaClO for 15min (use a shaker at 28°C 200r / min), sterilize with distilled water Rinse 3 times and transfer to a sterilized vessel, inoculate in 1 / 2MS medium. Cultivate in the dark at 25°C until germination, and then transfer to a light cultivation room. The seedling growth conditions are 25°C, 16h light / 8h dark, and light intensity 1800lx.
[0110] 2 Prepare explants and culture Agrobacterium
[0111] 6 days after the seeds germinate, cut off the cotyledons of the sterile seedlings with a knife. The cotyledons have a small petiole, and put them in the nursing medium KCMS for pre-cultivation for 1 day (avoid light, just overnight. If the nursing culture time is too long, it will easily lead to excessive infection. ). Pick a sin...
Embodiment 3
[0120] Construction of Tomato HsfA1a Gene Silencing Plants
[0121] The culture medium and antibiotic preparation method used in the present embodiment are as follows:
[0122] Preparation of YEB medium: 5g beef extract, 5g peptone, 1g yeast extract, 5g sucrose, 0.5g MgSO 4 ·7H 2 O, dilute to 1 L with distilled water, adjust the pH to 7.0, and autoclave at 121°C for 20 minutes for later use. Add 15g of agar powder per liter of YEB solid medium, and other components are the same as liquid medium.
[0123] Kanamycin: 50mg / mL, sterilized by filtration, and stored in aliquots at -20°C.
[0124] Rifampicin: Dissolve in 50mg / mL methanol, sterilize by filtration, store in aliquots at -20°C.
[0125] Gentamicin: 25 mg / mL was purchased from Shanghai Bioengineering Co., Ltd. and stored at -20°C.
[0126] Preparation of infection solution: 10 mM magnesium chloride, 10 mM MES, pH=5.7, add 150 μM / L acetosyringone when used.
[0127] The infection method is as follows:
[0128] (1) A...
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